BK Protein Expression Research Articles

A novel role of BK potassium channel activity in preventing the development of kidney fibrosis

Kidney fibrosis is a common characteristic of chronic kidney disease and while the large conductance voltage and calcium-activated potassium channel (BK) is widely expressed in kidneys, its role in kidney fibrosis is unknown. To evaluate this, we found that BK protein expression was decreased in the fibrotic kidneys. Accompanying this was increased fibrotic marker protein expression of fibronectin, vimentin and α-smooth muscle actin and increased mRNA expressions of fibronectin, α-smooth muscle actin, collagen III and collagen I. These changes occurred in the unilateral ureteral obstruction and folic acid models of fibrosis and were more pronounced in BK knockout than in wild-type mice. Activation of BK activity by chemical NS1619 or BMS191011 channel openers attenuated kidney fibrosis in these two models while protecting kidney function in wild-type mice. BK deficiency up-regulated transforming growth factor-β (TGF-β)/transcription factor Smad2/3 signaling in the fibrotic kidney, whereas activation of BK activity inhibited this signaling pathway both invivo and invitro. BK channel activation increased the degradation of TGF-β receptors induced by TGF-β1 invivo and invitro. Furthermore, in cell lines HK-2, NRK49, and NRK-52E, BK channel activation by NS1619 led to increased caveolae formation and facilitated localization of TGF-β receptors in the microdomains of lipid rafts. Thus, our data demonstrated that BK activation has an anti-fibrotic effect on kidney fibrosis by inhibiting the TGF-β signaling pathway through accelerating TGF-β receptor degradation via the caveolae route. Hence, our study provides innovative insight into BK as a potential therapeutic target for the treatment of kidney fibrosis.

Stimulatory Role of SPAK Signaling in the Regulation of Large Conductance Ca2+-Activated Potassium (BK) Channel Protein Expression in Kidney.

SPS1-related proline/alanine-rich kinase (SPAK) plays important roles in regulating the function of numerous ion channels and transporters. With-no-lysine (WNK) kinase phosphorylates SPAK kinase to active the SPAK signaling pathway. Our previous studies indicated that WNK kinases regulate the activity of the large-conductance Ca2+-activated K+ (BK) channel and its protein expression via the ERK1/2 signaling pathway. It remains largely unknown whether SPAK kinase directly modulates the BK protein expression in kidney. In this study, we investigated the effect of SPAK on renal BK protein expression in both HEK293 cells and mouse kidney. In HEK293 cells, siRNA-mediated knockdown of SPAK expression significantly reduced BK protein expression and increased ERK1/2 phosphorylation, whereas overexpression of SPAK significantly enhanced BK expression and decreased ERK1/2 phosphorylation in a dose-dependent manner. Knockdown of ERK1/2 prevented SPAK siRNA-mediated inhibition of BK expression. Similarly, pretreatment of HEK293 cells with either the lysosomal inhibitor bafilomycin A1 or the proteasomal inhibitor MG132 reversed the inhibitory effects of SPAK knockdown on BK expression. We also found that there is no BK channel activity in PCs of CCD in SPAK KO mice using the isolated split-open tubule single-cell patching. In addition, we found that BK protein abundance in the kidney of SPAK knockout mice was significantly decreased and ERK1/2 phosphorylation was significantly enhanced. A high-potassium diet significantly increased BK protein abundance and SPAK phosphorylation levels, while reducing ERK1/2 phosphorylation levels. These findings suggest that SPAK enhances BK protein expression by reducing ERK1/2 signaling-mediated lysosomal and proteasomal degradations of the BK channel.

14-3-3γ, a novel regulator of the large-conductance Ca2+-activated K+ channel.

14-3-3γ is a small protein regulating its target proteins through binding to phosphorylated serine/threonine residues. Sequence analysis of large-conductance Ca2+-activated K+ (BK) channels revealed a putative 14-3-3 binding site in the COOH-terminal region. Our previous data showed that 14-3-3γ is widely expressed in the mouse kidney. Therefore, we hypothesized that 14-3-3γ has a novel role in the regulation of BK channel activity and protein expression. We used electrophysiology, Western blot analysis, and coimmunoprecipitation to examine the effects of 14-3-3γ on BK channels both in vitro and in vivo. We demonstrated the interaction of 14-3-3γ with BK α-subunits (BKα) by coimmunoprecipitation. In human embryonic kidney-293 cells stably expressing BKα, overexpression of 14-3-3γ significantly decreased BK channel activity and channel open probability. 14-3-3γ inhibited both total and cell surface BKα protein expression while enhancing ERK1/2 phosphorylation in Cos-7 cells cotransfected with flag-14-3-3γ and myc-BK. Knockdown of 14-3-3γ by siRNA transfection markedly increased BKα expression. Blockade of the ERK1/2 pathway by incubation with the MEK-specific inhibitor U0126 partially abolished 14-3-3γ-mediated inhibition of BK protein expression. Similarly, pretreatment of the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of 14-3-3γ on BK protein expression. Furthermore, overexpression of 14-3-3γ significantly increased BK protein ubiquitination in embryonic kidney-293 cells stably expressing BKα. Additionally, 3 days of dietary K+ challenge reduced 14-3-3γ expression and ERK1/2 phosphorylation while enhancing renal BK protein expression and K+ excretion. These data suggest that 14-3-3γ modulates BK channel activity and protein expression through an ERK1/2-mediated ubiquitin-lysosomal pathway.

Dietary Na+ Depletion Transcriptionally Up‐regulates BK Channel Mediated K+ Secretion in Proximal Colon

BackgroundElectrogenic K+ secretion and H+‐K+‐ATPase‐mediated electroneutral K+ absorption occur in normal rat proximal and distal colon, respectively. Although both high‐K+ diet and dietary‐Na+ depletion induce K+ secretion in distal colon, only the dietary‐Na+ depletion has been shown to stimulate K+ secretion in proximal colon. Dietary‐Na+ depletion induced both intermediate conductance K+ (IK) and large conductance K+ (BK) channels mediated K+ secretion, while high‐K+ diet induced only the BK channel expression and activity in distal colon. Although K+ secretions are present, the molecular identity of K+ channels that mediate K+ secretion in normal and dietary‐Na+ depleted rat proximal colon is not known.HypothesisIt is hypothesized that dietary‐Na+ depletion may induce K+ secretion by stimulating IK and/or BK channel in proximal colon.AimThis study was initiated to identify the molecular identity of the dietary‐Na+ stimulated K+ secretion in rat proximal colon.MethodsNormal rats (Male Sprague‐Dawly; 200–225g) were fed either normal, high‐K+ or Na+‐depleted diet ad libitum for 6–7 days. Mucosal to serosal (m‐s) and serosal to mucosal (s‐m) 86Rb+ (K+ surrogate) fluxes were measured in stripped proximal colon epithelia mounted under voltage clamp condition. Net K+ fluxes were calculated by subtracting s‐m fluxes from m‐s fluxes. Positive and negative net K+ fluxes represent active absorption and secretion, respectively. IK and BK channel mRNA abundance and protein expression were determined by RT‐qPCR and western blot analyses, respectively. Cell and membrane specific IK and BK channel expressions were determined by immunofluorescence studies.ResultsSimilar to earlier demonstration, active K+ secretion is present in normal proximal colon. Dietary‐Na+ depletion, but not high‐K+ diet stimulated K+ secretion in proximal colon (Normal vs Na+‐depletion: 0.42 ± 0.06 vs 2.33 ± 0.16 μEq/cm2.h; p < 0.001). Mucosal iberiotoxin (IbTX; 10 μM; BK channel blocker) completely inhibited the K+ in normal, while it inhibited only 72% in Na+‐depleted rat proximal colon. Mucosal TRAM34 (10 μM; IK channel blocker) did not affect K+ secretion in normal, but it significantly (22%; p < 0.02) inhibited in Na+‐depleted rat colon. RT‐qPCR analyses revealed increased IK (2.2‐fold) and BK (6.2‐fold) specific mRNA abundance, while western blot analyses confirmed increased expression of IK and BK channel expression. Immunofluorescence studies established that in vitro aldosterone (1 μM) incubation increased both IK and BK protein expression in apical membranes of proximal crypt glands.ConclusionWe conclude that high (~1000 ng/ml; Na+‐depletion), but not low (~300 ng/ml; high‐K+ diet) blood aldosterone level stimulates IK/BK expression to increase K+ secretion in proximal colon.Support or Funding InformationThis study was supported by DK112085 and DK104791 from the National Institute of Health Diabetes and Digestive and Kidney Diseases.

G protein pathway suppressor 2 enhanced the renal large-conductance Ca2+-activated potassium channel expression via inhibiting ERK1/2 signaling pathway.

G protein pathway suppressor 2 (GPS2) is a multifunctional protein and transcriptional regulation factor that is involved in the G protein MAPK signaling pathway. It has been shown that the MAPK signaling pathway plays an important role in the regulation of renal large-conductance Ca2+-activated potassium (BK) channels. In this study, we investigated the effects of GPS2 on BK channel activity and protein expression. In human embryonic kidney (HEK) BK stably expressing cells transfected with either GPS2 or its vector control, a single-cell recording showed that GPS2 significantly increased BK channel activity ( NPo), increasing BK open probability ( Po), and channel number ( N) compared with the control. In Cos-7 cells and HEK 293 T cells, GPS2 overexpression significantly enhanced the total protein expression of BK in a dose-dependent manner. Knockdown of GPS2 expression significantly decreased BK protein expression, while increasing ERK1/2 phosphorylation. Knockdown of ERK1/2 expression reversed the GPS2 siRNA-mediated inhibition of BK protein expression in Cos-7 cells. Pretreatments of Cos-7 cells with either the lysosomal inhibitor bafilomycin A1 or the proteasomal inhibitor MG132 partially reversed the inhibitory effects of GPS2 siRNA on BK protein expression. In addition, feeding a high-potassium diet significantly increased both GPS2 and BK protein abundance in mice. These data suggest that GPS2 enhances BK channel activity and its protein expression by reducing ERK1/2 signaling-mediated degradation of the channel.

ASTROCYTE RESPONSE TO HYPERHOMOCYSTEINEMIA (HHCY)

Homocysteine (Hcy) is a non-protein forming amino acid involved in the production of both methionine and cysteine. Elevated Hcy can result from mutations in methylenetetrahydrofolate reductase (MTHFR) or cystathionine beta synthase (CBS) genes, as well as low levels of vitamins B6, B9, and B12. Elevated Hcy is termed hyperhomocysteinemia (HHcy). HHcy is a risk factor for vascular cognitive impairment and dementia (VCID), as well as Alzheimer's disease. The mechanism by which HHcy promotes VCID or AD remains unknown. Astrocytes play a crucial role in both potassium buffering and neurovascular coupling. The astrocytic end-feet essentially ensheath intraparenchymal blood vessels in the brain and express a variety of channels and markers indicative of their specialized functions. Channels enriched at the astrocytic end-feet include the aquaporin 4 water channel (AQP4), the inward rectifying potassium channel Kir4.1 and the calcium-dependent potassium channel BK. These channels are anchored at the astrocytic end-foot by dystrophin-1 (Dp71), as well as other anchoring proteins. To determine the cell specific effects of Hcy, C8-D1A astrocytes were treated with 50mM of Hcy for 24, 48, 72 and 96 hours. Gene expression for several astrocyte specific markers was then analyzed. To determine the in vivo effects of HHcy, wildtype mice were subject to a HHcy-inducing diet for a period of 6, 10 or 14 weeks. Tissue was then histologically examined for the astrocytic end-foot markers AQP4, Kir4.1, BK and dystrophin-1 (Dp71). Astrocytes treated with homocysteine exhibit an increased GFAP expression, suggesting homocysteine to be an inflammatory stimulus for astrocytes. While GFAP is increased, AQP4, Kir4.1, and BK expression levels were decreased. Our HHcy model displays significant astrocyctic end-foot disruptions. While AQP4 becomes dislocalized from the end-feet, Kir4.1 and BK protein expression decreases. In addition, the anchoring protein Dp71, also decreases. We examined mice subjected to the HHcy-inducing diet for 6, 10, and 14 weeks and find that end-disruption becomes progressively more severe as the mice continue the diet. This corresponds with progressively worse cognitive performance in the radial-arm water maze. HHcy results in the disruption of the astrocytic end-foot connection, which we hypothesize contributes to the cognitive decline observed in our HHcy mouse model.

BK K+ channel blockade inhibits radiation-induced migration/brain infiltration of glioblastoma cells.

Infiltration of the brain by glioblastoma cells reportedly requires Ca2+ signals and BK K+ channels that program and drive glioblastoma cell migration, respectively. Ionizing radiation (IR) has been shown to induce expression of the chemokine SDF-1, to alter the Ca2+ signaling, and to stimulate cell migration of glioblastoma cells. Here, we quantified fractionated IR-induced migration/brain infiltration of human glioblastoma cells in vitro and in an orthotopic mouse model and analyzed the role of SDF-1/CXCR4 signaling and BK channels. To this end, the radiation-induced migratory phenotypes of human T98G and far-red fluorescent U-87MG-Katushka glioblastoma cells were characterized by mRNA and protein expression, fura-2 Ca2+ imaging, BK patch-clamp recording and transfilter migration assay. In addition, U-87MG-Katushka cells were grown to solid glioblastomas in the right hemispheres of immunocompromised mice, fractionated irradiated (6 MV photons) with 5 × 0 or 5 × 2 Gy, and SDF-1, CXCR4, and BK protein expression by the tumor as well as glioblastoma brain infiltration was analyzed in dependence on BK channel targeting by systemic paxilline application concomitant to IR. As a result, IR stimulated SDF-1 signaling and induced migration of glioblastoma cells in vitro and in vivo. Importantly, paxilline blocked IR-induced migration in vivo. Collectively, our data demonstrate that fractionated IR of glioblastoma stimulates and BK K+ channel targeting mitigates migration and brain infiltration of glioblastoma cells in vivo. This suggests that BK channel targeting might represent a novel approach to overcome radiation-induced spreading of malignant brain tumors during radiotherapy.

Up-Regulatory Effects of Curcumin on Large Conductance Ca2+-Activated K+ Channels.

Large conductance Ca2+-activated potassium channels (BK) are targets for research that explores therapeutic means to various diseases, owing to the roles of the channels in mediating multiple physiological processes in various cells and tissues. We investigated the pharmacological effects of curcumin, a compound isolated from the herb Curcuma longa, on BK channels. As recorded by whole-cell patch-clamp, curcumin increased BK (α) and BK (α+β1) currents in transfected HEK293 cells as well as the current density of BK in A7r5 smooth muscle cells in a dose-dependent manner. By incubating with curcumin for 24 hours, the current density of exogenous BK (α) in HEK293 cells and the endogenous BK in A7r5 cells were both enhanced notably, though the steady-state activation of the channels did not shift significantly, except for BK (α+β1). Curcumin up-regulated the BK protein expression without changing its mRNA level in A7r5 cells. The surface expression and the half-life of BK channels were also increased by curcumin in HEK293 cells. These effects of curcumin were abolished by MG-132, a proteasome inhibitor. Curcumin also increased ERK 1/2 phosphorylation, while inhibiting ERK by U0126 attenuated the curcumin-induced up-regulation of BK protein expression. We also observed that the curcumin-induced relaxation in the isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. These results show that curcumin enhances the activity of the BK channels by interacting with BK directly as well as enhancing BK protein expression through inhibiting proteasomal degradation and activating ERK signaling pathway. The findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and the underlying mechanisms.